Circadian clocks are self-sustained biochemical oscillators that underlie daily rhythms of sleep/waking, metabolic activity, gene expression, and many other biological processes. Their properties include temperature compensation, a time constant of approximately 24 hours, and high precision. These properties are difficult to explain by known biochemical reactions. The ultimate explanation for the mechanism of these unusual oscillators will require characterizing the structures, functions, and interactions of the molecular components of circadian clocks. The simplest cells that are known to exhibit circadian phenomena are the prokaryotic cyanobacteria. Genetic and biochemical studies have identified three key clock proteins, KaiA, KaiB, and KaiC in the cyanobacterium Synechococcus elongatus. These three proteins plus ATP are competent to reconstitute a phosphorylation / dephosphorylation cycle in vitro that parallels the 24 hour cycles observed for global gene regulation in vivo. This in vitro circadian oscillator is the best available system for structural and biophysical analyses of a circadian clockwork. Preliminary electron microscopy (EM) data suggests that numerous and large conformational changes occur within the KaiA-KaiB-KaiC molecular oscillator, thus three-dimensional EM is well suited for structural analysis of this system. The specific aims of this proposal are 1) perform a cryoEM evaluation of three forms of the KaiB/KaiC complex with mutated forms of KaiC, and 2) determine a subnanometer resolution (<10[unreadable]) cryoEM structure of the chosen KaiB/KaiC complex. Characterizing the molecular mechanisms that allow a circadian clockwork to oscillate with a 24 hour cycle is essential for understanding circadian rhythms in cyanobacteria to humans. Detailed structural analysis of the core oscillator from cyanobacteria will provide the mechanisms underlying the controlled protein-protein associations that appear to drive this unique clockwork. The long-term goal of this proposal is to apply hybrid methods including three-dimensional EM to characterize the structure and function of supramolecular protein complexes formed during the cyanobacterial circadian oscillation cycle.